The sphingolipid ceramide is a potent tumor suppressor that contributes to the promotion of apoptosis and autophagy. Management of these responses in cancer cells is dependent on the dynamic balance between ceramide which is pro-apoptotic and its metabolites, which can promote cell survival. Upregulated glycosylation of ceramide, a major pathway contributing to ceramide detoxification, limits the valuable tumor suppressor effects of ceramide. This proposal will focus on reducing ceramide glycosylation with the goal of devising novel ceramide therapies for treatment of acute myeloid leukemia (AML), the most common type of leukemia in adults. Upregulated ceramide catabolism is associated with multidrug resistance in leukemia. In our study we will use ceramide exogenously in the form of short-chain, cell-permeable C6-ceramide (C6-cer) nanoliposomes, a system that offers advantages over other delivery systems or the use of natural ceramide, which due to solubility and transport limitations, cannot be administered exogenously. Our objective is to develop innovative approaches for treating AML, and this will be achieved by delivering C6-cer in conjunction with adjuvants that will attenuate metabolism and thereby amplify ceramide-driven cell death. To attenuate C6-cer metabolism, we will employ P-glycoprotein (P-gp) antagonists as adjuvants. We hypothesize that intracellular P-gp, because of its role in glycolipid trafficking, will be a highly effective target for attenuating C6-cer metabolism.. We also hypothesize that P-gp antagonists will increase the intracellular levels of both C6-cer and long-chain ceramides and magnify end-point responses (apoptosis, autophagy, cell cycle arrest). Because of the major role that P-gp plays in regulating the metabolism of short-chain ceramides, this project hallmarks a shift in clinical strategy by introducing the use of P-gp antagonists as modulators of ceramide metabolism and enhancers of ceramide cytotoxicity, an activity divorced from the much investigated chemotherapy effluxing/multidrug resistance protein we all know. To make nanoliposomal C6-cer more effective as a therapeutic in AML, the following aims will be pursued: 1) Determine the influence of P-gp expression on C6-cer cytotoxicity and metabolism in AML cell models. Understanding the relationship of P- gp and cellular response to C6-cer is critical for optimizing ceramide-based therapeutics. 2) Determine the effect of inhibiting C6-cer catabolism on cytotoxic response to C6-cer in defined AML cell lines and patient cells. We hypothesize that blocking C6-cer catabolism, including conversion to sphingomyelin, will synergistically amplify cytotoxicity. 3) Elucidate the mechanism of cell death and the signaling events associated with cytotoxicity of combination C6-cer/P-gp antagonists in AML cells. 4) Determine the effect of adjuvant inhibitors on response to C6-cer therapy in in vivo AML mouse models. Dysfunctional ceramide metabolism promotes resistance to apoptosis. We expect to discover that partnering nanoliposomal C6-cer with P-gp antagonists (that block ceramide catabolism) will be a novel, innovative, non-toxic treatment for AML.